In article <3agrfu$kg1 at mule.fhcrc.org>, Terese Maltzman
<tmaltzma at fred.fhcrc.org> wrote:
> Does anyone out there have a "tried and true", consistent protocol for
> extracting DNA from paraffin-embedded tissue sections? Most of my
> sections have been formalin fixed. I am trying to amplify products that
> are 200-350 bp long.
>> thanks
>> Terese Maltzman
> FHCRC
We have developed a method that really is "tried and true,"and is simple
to boot. As a surgical pathologist doing molecular biology, I have
extracted probably DNA from 1000's of paraffin sections. We compared
methods and fiddled and this is the technique we eventually came up with:
(from: Goldblum JR, Bartos RE, Carr KA, Frank TS: Hepatitis B and
alterations of the p53 tumor suppressor gene in hepatocellular carcinoma.
Am J Surg Pathol 1993, 17: 1244-1251.)
Unstained and unheated (important) tissue sections are scraped off with
a disposable razor blade and deparaffinized with 100 µl of xylene in a 1.7
ml nonsiliconized microcentrifuge tube. An equal volume of 100% ethanol is
added and the samples are pelleted (10 min in a countertop
microcentrifuge), dried under vacuum or in a 55° oven for 20 min (not too
much), then digested overnight with 200 ng/µl Proteinase K in 100µl of
50 mM tris pH 8.3 at 37°C (no detergents, especially ionic detergents).
Samples are then boiled for 8 min, placed on ice, and spun 5 minutes to
pellet out debris. That's it! Also, we use a ³mock-extraction² negative
control containing only reagents.
To evaluate this, we performed serial dilutions of DNA extracted from
paraffin sections by a variety of different techniques. DNA was extracted
either by proteinase K digestion with ionic detergents followed by phenol
chloroform extraction, proteinase K digestion without detergents followed
by boiling, sonication with proteinase K followed by boiling, or direct
boiling alone. The technique cited above gave the best yield: to an
equivalent of one 40X microscopic field of tissue. Proteinase K digestion
with detergents followed by phenol-chloroform extraction was no better
than simply boiling the paraffin section in water! Although the
efficiency of PCR will vary with fixative and size of the amplified
fragment, proteinase K digestion of even small paraffin tissue sections
results in sufficient DNA for successful PCR amplification from
single-copy genes and visualization of PCR product on an ethidium-stained
agarose gel after 35 cycles of PCR.
For boiling the samples we use a standard hot-pot (one that does NOT
shut off when the water starts to boil, of course) and an immersible metal
boiling rack that contains 16 places for 1.5ml tubes ($35.00), catalog #
142350 from RPI, 410 North Business Center Dr., Mount Prospect, IL 60056
(USA)/ 800-323-9814.
Let me know how it works!
Tom Frank
tom.frank at med.umich.edu
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